Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A novel bacteriophage defense system, based on an inducible suicide gene, was challenged with a lactococcal bacteriophage to investigate the potential for phage adaptation. The defense system was encoded by pTRK414H, a high-copy-number replicon encoding a tightly regulated phi 31p trigger promoter fused to the lethal LlaIR+ restriction endonuclease cassette. Repeated transfers of Lactococcus lactis NCK690(pTRK414H) in the presence of phi 31 selected for phage phi 31 derivatives which were markedly less sensitive to phi 31p-LlaIR(+)-encoded restriction than the parental phage, phi 31. The efficiency of plaquing (EOP) on L. lactis NCK690(pTRK414H) was 10(-4) for phi 31 versus 0.4 for the derived phages. The mutant phages remained fully sensitive to LlaIR+ restriction, suggesting an alteration in the recognition or firing of the phi 31p promoter. Sequencing over the promoter region in four mutant phages revealed the identical C-to-A transversion, generating a Phe-to-Leu substitution, in a transcriptional activator of the phi 31p promoter, designated ORF2. The mutant phages were analyzed for their ability to induce the native phi 31p promoter element fused to a lacZst reporter gene. Compared to the parental phage, phi 31, lower levels of beta-galactosidase activity were induced throughout the lytic cycle, indicating that the strength at which the mutant phages activated the phi 31p promoter was altered. Based on these observations, improvements were made in promoter strength and restriction activity in an attempt to elevate the effectiveness of the phage-triggered suicide system. When the phi 31p-LlaIR+ cassette was paired with other abortive defense systems, Per31 and AbiA, the EOP of phi 31 was reduced to < 10(-10) and the level of phage in the culture was lowered below the detection limits of the assay.
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PMID:Bacteriophage-triggered defense systems: phage adaptation and design improvements. 936 24

The homogenate from Caco-2 cells of day 13 or 15 after subculturing had high omega-hydroxylation activity of docosahexaenoic acid (22:6(n-3)) or arachidonic acid (20:4(n-6)). Activity, maximal at pH 8.0, was inhibited in the presence of CO or metyrapone and in the absence of NADPH. Omega-hydroxylation activity of lauric acid in the homogenate was not detected. Apparent Michaelis constant (Km) values for 22:6(n-3) and 20:4(n-6) were found to be 4 and 7 microM. Omega-hydroxylation activity considerably increased with growth up to day 13 and then decreased until day 20 even though alkaline phosphatase (ALP) and leucine-aminopeptidase (LAP) activity increased with growth to day 20. Metyrapone in cultures caused omega-hydroxylation, ALP and LAP activity to decrease, while sodium butyrate dose-dependently increased that of omega-hydroxylation, ALP and an endogenous endonuclease and decreased lactate dehydrogenase (LDH) activity in culture medium. The omega-hydroxylation system thus appears quite likely to be associated with cytochrome P450, differentiation and/or apoptosis rather than cytotoxic cell death of Caco-2 cells. Substrate specificity, however, differed from that of human cytochrome P450 4A11.
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PMID:Docosahexaenoic/arachidonic acid omega-hydroxylation system and differentiation in the human colonic adenocarcinoma cell line, Caco-2. 946 91

The 9B, 123, 788/8 Pseudomonas syringae phages were investigated. PAAG electrophoretic profiles of phage proteins were identical for all three phages except the minor polypeptide having molecular weight 35,000 Da. The band corresponding to this protein was present only in 9B and 788/8 phage protein profiles. Amino acid composition of phage proteins varied insignificantly showing prevalence of Asp, Glu, Ala, Leu. Phage DNA fragments electrophoresis, carried out after processing with specific endonuclease Hind III, made it possible to evaluate common restriction sites in phage genomes. Genome molecular weight was equal to 15 mda for 9B phage and to 14 mDa for 123 and 788/8 phages. The analysis of phage growth cycle showed that latent period consisted of 50 min at 20 degrees C and the yield equalled to 70 virions per infected bacterium cell. The similarity of the phages' features suggests their broad spreading in the environment.
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PMID:[The properties of the proteins and nucleic acids of 3 Pseudomonas syringae phages]. 948 14

Nae I protein was originally isolated for its restriction endonuclease properties. Nae I was later discovered to either relax or cleave supercoiled DNA, depending upon whether Nae I position 43 contains a lysine (43K) or leucine (43L) respectively. Nae I-43K DNA relaxation activity appears to be the product of coupling separate endonuclease and ligase domains within the same polypeptide. Whereas Nae I relaxes supercoiled DNA like a topoisomerase, even forming a transient covalent intermediate with the substrate DNA, Nae I shows no obvious sequence similarity to the topoisomerases. To further characterize the topoisomerase activity of Nae I, we report here that Nae I-43K changes the linking number of a single negatively supercoiled topoisomer of pBR322 by units of one and therefore is a type I topoisomerase. Positively supercoiled pBR322 was resistant to Nae I-43K. At low salt concentration Nae I-43K was processive; non-saturating amounts of enzyme relaxed a fraction of the DNA. At high salt concentration the same non-saturating amounts of Nae I-43K partially relaxed all the DNA in a step-wise fashion to give a Gaussian distribution of topoisomers, demonstrating a switch from a processive to a distributive mode of action. Nae I-43K decatenated kinetoplast DNA containing nicked circles, implying that Nae I-43K can cleave opposite a nick. The products of the reaction are decatenated nicked circles under both processive and distributive conditions. The behavior of Nae I-43K is consistent with that of a prokaryotic type I topoisomerase.
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PMID:Step-wise DNA relaxation and decatenation by NaeI-43K. 958 Jun 89

Long interspersed elements, or LINEs, are retrotransposons that move via an RNA intermediate. In mice, one polymorphic variant of L1 has amplified relatively recently, giving rise to the A-type subfamily in species belonging to the genus and subgenus Mus. Retrotransposition of LINE-1 (L1) requires the function of the L1-encoded reverse transcriptase that is produced from open reading frame 2 (ORF2). Here, we employ a convenient yeast genetic assay to determine the reverse transcriptase activity of the ORF2 obtained from three A-type L1 elements: one, a cDNA from the RNA in ribonucleoprotein particles; another with a purported inactivating mutation; and the third, a hypothetical ancestral construct. Because there are no examples of A-type elements that have transposed recently to inactivate a gene, this assay is the first step towards demonstrating the functional capability of mouse A-type LINE-1 elements. One of the three elements was believed to have been inactivated during evolution by the substitution of leucine for a highly conserved phenylalanine or tryptophan residue among known reverse transcriptases. This mutation did not inactivate the L1 reverse transcriptase in the yeast assay; thus, all three of the elements tested encoded reverse transcriptase activity. We further examined the minimal reverse transcriptase domain within ORF2 by creating a series of deletions. The results demonstrate that removal of the L1 endonuclease domain from the N-terminal region of ORF2 does not affect reverse transcriptase activity as determined by this assay, and that approximately half of the ORF2 coding sequence from mouse A-type L1 elements is required for functional reverse transcriptase.
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PMID:Functional reverse transcriptases encoded by A-type mouse LINE-1: defining the minimal domain by deletion analysis. 966 81

To improve the production of D-amino acids using an immobilized N-carbamyl-D-amino acid amidohydrolase, the enzyme gene of Agrobacterium sp. KNK712 was mutagenized randomly to increase its thermostability. The gene was inserted into M13mp19, mutagenized with hydroxylamine, ligated into pUC19 after restriction endonuclease digestion, and then used to transform Escherichia coli. The resultant transformants were screened by a newly developed colorimetric enzyme assay method, and the candidate colonies corresponding to red spots were separated from the master plates. Using cell-free extracts of these clones, the properties of the enzymes produced were investigated, it being proved that these enzymes had almost the same activity and improved thermostability by about 5 degrees C compared with those of the native enzyme. As found on enzyme gene analysis of these mutants, the 57th amino acid, histidine, of the enzyme was changed to tyrosine, or the 203rd amino acid, proline, to leucine or serine.
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PMID:Increase in thermostability of N-carbamyl-D-amino acid amidohydrolase on amino acid substitutions. 980 66

Ribonuclease P activity from infusoria Tetrahymena pyriformis has been isolated and purified more than 1000-fold over cytosol crude extract. Purified tRNA 5' endonuclease processes in vitro heterologous substrates, precursors of the human tRNA(Tyr) and Drosophila melanogaster tRNA(Leu), exactly at the 5' end of the mature molecules. The activity was abolished by micrococcal nuclease and protease treatment indicating that both RNA and protein components are essential for its activity. The most abundant polypeptides in the purified enzyme fractions have molecular masses of about 100, 44 and 35 kDa. The enzyme requires divalent cations for its activity and shows optimal activity in the presence of the low concentrations of the monovalent salts. Substrate structural requirements for the purified enzyme were analyzed with different tRNA precursor models. The analysis of the derivatives of tRNA(Leu) precursors with altered aminoacyl stem structures reveals that end of the stem is important for substrate 5' end processing with purified enzyme.
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PMID:Partial characterization of the ribonuclease P from Tetrahymena pyriformis. 981 Apr 66

Despite a wide interindividual variation of cytochrome P-450 1A2 (CYP1A2) activity, genetic polymorphism of CYP1A2 has not been reported. By amplification of exons of CYP1A2 by polymerase chain reaction in eight Chinese subjects, the polymerase chain reaction products were directly sequenced. One subject showed heterozygous C2866-->G (Phe21-->Leu) polymorphism. DNA from 157 Chinese subjects (104 polychlorinated biphenyl-exposed subjects and 53 control subjects) was screened for polymorphism by single-strand conformation polymorphism method and MboII endonuclease digestion. Only 1 of 157 samples showed another heterozygous C2866-->G mutation. The subject was previously exposed to polychlorinated biphenyl and showed a value of 3.5% in the caffeine breath test. The value is not significantly higher than the mean value of polychlorinated biphenyl-exposed subjects (3.12 +/- 0.29%, mean +/- S.E.M.). The incidence of the point mutation in these Chinese subjects is less than 1%. The prevalence of the F21L mutation in other ethnic groups and its effect on the metabolic activity of CYP1A2 remain to be further evaluated.
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PMID:Detection of a novel cytochrome P-450 1A2 polymorphism (F21L) in Chinese. 988 16

Background: Several mutations in mitochondrial DNA (mtDNA) are associated with the syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). The "common" MELAS mutation, A3243G in the tRNA leucine (UUR) gene, affects approximately 80% of cases and is associated with respiratory chain complex I deficiency. Methods and Results: The A3243G mutation creates an ApaI restriction endonuclease site and can be detected by polymerase chain reaction (PCR) amplification of a region of mtDNA containing nt 3243, followed by ApaI digestion and electrophoretic analysis of the resulting fragments. Analysis of mtDNA from a child with complex I deficiency indicated the presence of the mutation homoplasmically in heart, liver, and skeletal muscle. Sequencing revealed only normal tRNA leucine (UUR) sequence, and a novel variant at nt 3426 in the ND1 subunit of complex I, which creates an ApaI site. ApaI digestion results in fragments of similar size to those found in patients with the A3243G mutation. Conclusions: A novel variant at nt 3426 of mtDNA creates an ApaI site and can potentially cause a false-positive result for the presence of the A3243G mutation. Given the highly polymorphic nature of mtDNA, care must be exercised in choosing primers for restriction endonuclease-based diagnostic tests for point mutations, and confirmation of a mutation by an independent method is recommended.
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PMID:A False-Positive Diagnosis for the Common MELAS (A3243G) Mutation Caused by a Novel Variant (A3426G) in the ND1 Gene of Mitochondria DNA. 1008 79

Following random mutagenesis of the Eco RV endonuclease, a high proportion of the null mutants carry substitutions at Gln69. Such mutants display reduced rates for the DNA cleavage step in the reaction pathway, yet the crystal structures of wild-type Eco RV fail to explain why Gln69 is crucial for activity. In this study, crystal structures were determined for two mutants of Eco RV, with Leu or Glu at residue 69, bound to specific DNA. The structures of the mutants are similar to the native protein and no function can be ascribed to the side chain of the amino acid at this locus. Instead, the structures of the mutant proteins suggest that the catalytic defect is due to the positioning of the main chain carbonyl group. In the enzyme-substrate complex for Eco RV, the main chain carbonyl of Gln69 makes no interactions with catalytic functions but, in the enzyme-product complex, it coordinates a metal ion bound to the newly liberated 5'-phosphate. This re-positioning may be hindered in the mutant proteins. Molecular dynamics calculations indicate that the metal on the phosphoryl oxygen interacts with the carbonyl group upon forming the pentavalent intermediate during phosphodiester hydrolysis. A main chain carbonyl may thus play a role in catalysis by Eco RV.
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PMID:Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group. 1044 31


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